Domain formation in a fluid mixed lipid bilayer modulated through binding of the C2 protein motif

The role and mechanism of formation of lipid domains in a functional membrane have generally received limited attention. Our approach, based on the hypothesis that thermodynamic coupling between lipid-lipid and protein-lipid interactions can lead to domain formation, uses a combination of an experimental lipid bilayer model system and Monte Carlo computer simulations of a simple model of that system. The experimental system is a fluid bilayer composed of a binary mixture of phosphatidylcholine (PC) and phosphatidylserine (PS), containing 4% of a pyrene-labeled anionic phospholipid. Addition of the C2 protein motif (a structural domain found in proteins implicated in eukaryotic signal transduction and cellular trafficking processes) to the bilayer first increases and then decreases the excimer/monomer ratio of the pyrene fluorescence. We interpret this to mean that protein binding induces anionic lipid domain formation until the anionic lipid becomes saturated with protein. Monte Carlo simulations were performed on a lattice representing the lipid bilayer to which proteins were added. The important parameters are an unlike lipid-lipid interaction term and an experimentally derived preferential protein-lipid interaction term. The simulations support the experimental conclusion and indicate the existence of a maximum in PS domain size as a function of protein concentration. Thus, lipid-protein coupling is a possible mechanism for both lipid and protein clustering on a fluid bilayer. Such domains could be precursors of larger lipid-protein clusters ('rafts'), which could be important in various biological processes such as signal transduction at the level of the cell membrane.     

Effects of normal alcohols and isoflurane on lipid headgroup dynamics in nicotinic acetylcholine receptor-rich lipid vesicles

The trend of evidence suggests that general anesthetics act directly on proteins in the neural membrane. However, the fact that the functions of nicotinic acetylcholine receptor (sodium permeability, desensitization rate) are modulated by the composition of the membrane in which it is reconstituted has been thought to be a result of the variation of interactions between acetylcholine receptor and membrane. In this study, protein-lipid interaction at the level of the lipid headgroup was investigated using electron paramagnetic resonance (EPR) and headgroup spin label. Lipid headgroup mobility was evaluated with rotational correlation time from the EPR spectrum. Protein-lipid interaction at headgroup depth was demonstrated from the motionally restricted component of the spectrum. Rotational correlation time increased to 13 ns from 7 ns due to protein-lipid interaction. The effect of anesthetic (ethanol, 1-hexanol, and isoflurane) on protein-lipid interaction was investigated, and the correlation time was 13 ns. It is concluded that the anesthetics used in this study did not alter protein-lipid interaction at the level of the lipid headgroup, so far as observed by rotational correlation time, without excluding the possibility that anesthetics that perturb protein-lipid interactions modulate receptor functions via this mechanism.     

Lipid enrichment and selectivity of integral membrane proteins in two-component lipid bilayers

A model recently used to study lipid-protein interactions in one-component lipid bilayers (Sperotto and Mouritsen, 1991 a, b) has been extended in order to include two different lipid species characterized by different acyl-chain lengths. The model, which is a statistical mechanical lattice model, assumes that hydrophobic matching between lipid-bilayer hydrophobic thickness and hydrophobic length of the integral protein is an important aspect of the interactions. By means of Monte Carlo simulation techniques, the lateral distribution of the two lipid species near the hydrophobic protein-lipid interface in the fluid phase of the bilayer has been derived. The results indicate that there is a very structured and heterogeneous distribution of the two lipid species near the protein and that the protein-lipid interface is enriched in one of the lipid species. Out of equilibrium, the concentration profiles of the two lipid species away from the protein interface are found to develop a long-range oscillatory behavior. Such dynamic membrane heterogeneity may be of relevance for determining the physical factors involved in lipid specificity of protein function.     

Quantitative characterization of the lateral distribution of membrane proteins within the lipid bilayer.

The dependence of the lateral distribution of membrane proteins on the size, protein/lipoid molar ratio, and the magnitude of the interaction potentials has been investigated by computer modeling protein-lipid distributions with Monte Carlo calculations. These results have allowed us to develop a quantitative characterization of the distribution of membrane proteins and to correlate these distributions with experimental observables. The topological arrangement of protein domains, protein plus annular lipid domains, and free lipid domains is described in terms of radial distribution, pair connectedness, and cluster distribution functions. The radial distribution functions are used to measure the distribution of intermolecular distances between protein molecules, whereas the pair connectedness functions are used to estimate the physical extension of compositional domains. It is shown that, at characteristic protein/lipid molar ratios, previously isolated domains become connected, forming domain networks that extend over the entire membrane surface. These changes in the lateral connectivity of compositional domains are paralleled by changes in the calculated lateral diffusion coefficients and might have important implications for the regulation of diffusion controlled processes within the membrane.     

Regulation of channel function due to physical energetic coupling with a lipid bilayer

Regulation of membrane protein functions due to hydrophobic coupling with a lipid bilayer has been investigated. An energy formula describing interactions between lipid bilayer and integral ion channels with different structures, which is based on the screened Coulomb interaction approximation, has been developed. Here the interaction energy is represented as being due to charge-based interactions between channel and lipid bilayer. The hydrophobic bilayer thickness channel length mismatch is found to induce channel destabilization exponentially while negative lipid curvature linearly. Experimental parameters related to channel dynamics are consistent with theoretical predictions. To measure comparable energy parameters directly in the system and to elucidate the mechanism at an atomistic level we performed molecular dynamics (MD) simulations of the ion channel forming peptide–lipid complexes. MD simulations indicate that peptides and lipids experience electrostatic and van der Waals interactions for short period of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides (in ion channel) and lipids (in lipid bilayer) due to mainly their charge properties. The results of in silico MD studies taken together with experimental observable parameters and theoretical energetic predictions suggest that the peptides induce ion channels inside lipid membranes due to peptide–lipid physical interactions. This study provides a new insight helping better understand of the underlying mechanisms of membrane protein functions in cell membrane leading to important biological implications.     

Statistical thermodynamic analysis of peptide and protein insertion into lipid membranes.

A statistical thermodynamic approach is used to analyze the various contributions to the free energy change associated with the insertion of proteins and protein fragments into lipid bilayers. The partition coefficient that determines the equilibrium distribution of proteins between the membrane and the solution is expressed as the ratio between the partition functions of the protein in the two phases. It is shown that when all of the relevant degrees of freedom (i.e., those that change their character upon insertion into the membrane) can be treated classically, the partition coefficient is fully determined by the ratio of the configurational integrals and thus does not involve any mass-dependent factors, a conclusion that is also valid for related processes such as protein adsorption on a membrane surface or substrate binding to proteins. The partition coefficient, and hence the transfer free energy, depend only on the potential energy of the protein in the membrane. Expressing this potential as a sum of a "static" term, corresponding to the equilibrium (minimal free energy) configuration of the protein in the membrane, and a "dynamical" term representing fluctuations around the equilibrium configuration, we show that the static term contains the "solvation" and "lipid perturbation" contributions to the transfer free energy. The dynamical term is responsible for the "immobilization" free energy, reflecting the loss of translational and rotational entropy of the protein upon incorporation into the membrane. Based on a recent molecular theory of lipid-protein interactions, the lipid perturbation and immobilization contributions are then expressed in terms of the elastic deformation free energy resulting from the perturbation of the lipid environment by the foreign (protein) inclusion. The model is formulated for cylindrically shaped proteins, and numerical estimates are given for the insertion of an alpha-helical peptide into a lipid bilayer. The immobilization free energy is shown to be considerably smaller than in previous estimates of this quantity, and the origin of the difference is discussed in detail.     

Direct observation of alpha-lactalbumin,adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures

The interaction between proteins and membranes is of great interest in biomedical and biotechnological research for its implication in many functional and dysfunctional processes. We present an experimental study on the interaction between model membranes and alpha-lactalbumin (α-La). α-La is widely studied for both its biological function and its anti-tumoral properties. We use advanced fluorescence microscopy and spectroscopy techniques to characterize α-La-membrane mechanisms of interaction and α-La-induced modifications of membranes when insertion of partially disordered regions of protein chains in the lipid bilayer is favored. Moreover, using fluorescence lifetime imaging, we are able to distinguish between protein adsorption and insertion in the membranes. Our results indicate that, upon addition of α-La to giant vesicles samples, protein is inserted into the lipid bilayer with rates that are concentration-dependent. The formation of heterogeneous hybrid protein-lipid co-aggregates, paralleled with protein conformational and structural changes, alters the membrane structure and morphology, leading to an increase in membrane fluidity.     

Cytochrome C interaction with cardiolipin/phosphatidylcholine model membranes: effect of cardiolipin protonation

Resonance energy transfer between anthrylvinyl-labeled phosphatidylcholine as a donor and heme moiety of cytochrome c (cyt c) as an acceptor has been employed to explore the protein binding to model membranes, composed of phosphatidylcholine and cardiolipin (CL). The existence of two types of protein-lipid complexes has been hypothesized where either deprotonated or partially protonated CL molecules are responsible for cyt c attachment to bilayer surface. To quantitatively describe cyt c membrane binding, the adsorption model based on scaled particle and double layer theories has been employed, with potential-dependent association constants being treated as a function of acidic phospholipid mole fraction, degree of CL protonation, ionic strength, and surface coverage. Multiple arrays of resonance energy transfer data obtained under conditions of varying pH, ionic strength, CL content, and protein/lipid molar ratio have been analyzed in terms of the model of energy transfer in two-dimensional systems combined with the adsorption model allowing for area exclusion and electrostatic effects. The set of recovered model parameters included effective protein charge, intrinsic association constants, and heme distance from the bilayer midplane for both types of protein-lipid complexes. Upon increasing CL mole fraction from 10 to 20 mol % (the value close to that characteristic of the inner mitochondrial membrane), the binding equilibrium dramatically shifted toward cyt c association with partially protonated CL species. The estimates of heme distance from bilayer center suggest shallow bilayer location of cyt c at physiological pH, whereas at pH below 6.0, the protein tends to insert into membrane core.     

The electrostatics of lipid surfaces

Charged lipids constitute a substantial fraction of all membrane lipids. Their charges vary in quantity and distribution within their headgroup regions. In long range interactions, their charges' value and electrostatic potential in the vicinity of the membrane surface can be approximated by the Guy-Chapman theory. This theory treats the interface as a charged structureless plain surrounded by uniform environments. However, if one considers intermolecular interactions, such assumptions need to be revised. The interface is in reality a thick region containing the residual charges of lipid headgroups. Their arrangement depends on the type of lipid present in the membrane. The variety of lipids and their biological functions suggests that charge distribution determines the extent and type of interaction with surface associated molecules. Numerous examples show that protein behavior at the lipid bilayer surface is determined by the type of lipid present, indicating protein specificity towards certain surface locations and local properties (determined by lipid composition) of a particular type. Such specificity is achieved by a combination of electrostatic, hydrophobic and enthropic effects. Comparing lipid biological activity, it can be stated that residual charge distribution is one of the factors of intermolecular recognition leading to the specific interaction of lipid molecules and selected proteins in various processes, particularly those involved with signal transduction pathways. Such specificity enables a variety of processes occurring simultaneously on the same membrane surface to function without cross-reaction interference.     

Setting up and optimization of membrane protein simulations

In this paper we describe a method for setting up an atomistic simulation of a membrane protein in a hydrated lipid bilayer and report the effect of differing electrostatic parameters on the drift in the protein structure during the subsequent simulation. The method aims to generate a suitable cavity in the interior of a lipid bilayer, using the solvent-accessible surface of the protein as a template, during the course of a short steered molecular dynamics simulation of a solvated lipid membrane. This is achieved by a two-stage process: firstly, lipid molecules whose headgroups are inside a cylindrical volume equivalent to that defined by the protein surface are removed; then the protein-lipid interface is optimized by applying repulsive forces perpendicular to the protein surface, and of gradually increased magnitude, to the remaining lipid atoms inside the volume occupied by the protein surface until it is emptied. The protein itself may then be inserted. Using the bacterial membrane proteins KcsA and FhuA as test cases, we show how the method achieves the formation of a suitable cavity in the interior of a dimyristoylphosphatidylcholine lipid bilayer without perturbing the configuration of the non-interfacial regions of the previously equilibrated lipid bilayer, even in cases of membrane proteins with irregular geometrical shapes. In addition, we compare subsequent simulations in which the long-range electrostatic interactions are treated via either a cut-off or a particle-mesh Ewald method. The results show that the drift from the initial structure is less in the latter case, especially for KcsA and for the non-core secondary structural elements (i.e. surface loops) of both proteins.     

Association of the internal membrane protein with the lipid bilayer in influenza virus. A study with the fluorescent probe 12-(9-anthroyl)-stearic acid

The lipid fluorescent probe 12-(9-anthroyl)-stearic acid was introduced into the lipid bilayer of influenza virus particles. Fluorescent energy transfer was observed from the viral protein to the probe. This transfer persisted after removal of the glycoprotein spikes which cover the outside of the viral particle, demonstrating that the energy donor was an internal protein. It was concluded that the energy donor was the non-glycosylated membrane protein (M protein), the major protein component of the spikeless particle. Analysis of the emission spectrum of the spikeless particle excited at 275 nm shows that a substantial portion of the fluorescence arises from tyrosine residues, in contrast to most other proteins which contain both tryptophan and tyrosine.It is suggested that the donor residue(s) are located no more than 11 Å exterior to the bilayer surface, and that a portion of the M protein may penetrate into the bilayer.     

A liquid diffraction analysis of sarcoplasmic reticulum. I. Compositional variation.

Intensities of x-ray scattering from a series of fragmented rabbit muscle sarcoplasmic reticulum (SR) samples have been measured over the range x = 0.05 to s = 0.25. By varying the relative concentrations of lipid and protein (chiefly the Mg++-dependent, Ca++- stimulated ATPase) in the membranes of this series, and by employing methods of analysis appropriate to the scattering from binary liquid mixtures, we have identified the separable contributions of protein and lipid, and the protein-lipid interaction contributions to the total scattering profiles. The shape of the protein term is consistent with scattering from a cylindrical ATPase particle 142 A in length and 35 A in diameter. These data imply that the dominant ATPase species is monomeric. The protein-lipid interaction term has been analyzed by a novel treatment based on a determination of the pair correlation function between the electrons of the protein molecule with the electrons of the lipid bilayer in terms of the asymmetry of the transbilayer disposition of the protein. Applied to our results, the analysis indicates a fully asymmetric disposition of ATPase, in which one end of the molecule is contiguous with either the lumenal or cytoplasmic surface of the bilayer.     

Human platelet P-235, a talin-like actin binding protein, binds selectively to mixed lipid bilayers

The interaction of platelet talin (P-235) with mixtures of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylserine (DMPS) as well as with pure lipids was studied in reconstituted lipid bilayers. Incorporation of platelet talin into vesicles was achieved by self-assembly during cycles of freeze-thawing of co-dispersions containing vesicles and the purified protein. The yield of protein incorporation as a function of lipid composition was determined by measuring the protein/lipid ratio using protein assay, phosphate determination and gel electrophoresis in parallel. Protein-lipid interactions are monitored by high sensitive differential scanning calorimetry (DSC) measuring (i) the shifts of transition states delta Ts* and delta Tl*, where Ts represents the solidus line, the onset of lipid chain melting, and Tl the liquidus line, the endpoint of chain melting, and (ii) the heats of transition. Cytoplasmic talin differs from a membrane bound form by its ability and mode of lipid interaction. The latter partially penetrates into the hydrophobic region of the bilayer, which renders a low incorporation rate even into neutral lipids. This interaction is greatly enhanced in the presence of charged lipids: a marked shift of Tl occurs due to a selective electrostatic interaction of the protein with the membrane surface. Evidence for a selective binding is also provided by Fourier transform infrared spectroscopy (FTIR). Right-side-out oriented platelet talin can be cleaved by proteinases, which truncate the extrinsic electrostatic binding domain but not the hydrophobic. In addition, reconstituted platelet talin, like in vivo, can be cleaved by thrombin. The interaction of cytoplasmic platelet talin with lipid bilayers is purely electrostatic. Our data suggest that protein reconstitution by freeze-thawing is an equilibrium process and that the protein distribution between the membrane and water is determined by the Nernst distribution law. Consequently, the work of protein transfer from water into the bilayer can be measured as a function of charged lipids.     

Thermosensing via transmembrane protein–lipid interactions

Cell membranes are composed of a lipid bilayer containing proteins that cross and/or interact with lipids on either side of the two leaflets. The basic structure of cell membranes is this bilayer, composed of two opposing lipid monolayers with fascinating properties designed to perform all the functions the cell requires. To coordinate these functions, lipid composition of cellular membranes is tailored to suit their specialized tasks. In this review, we describe the general mechanisms of membrane–protein interactions and relate them to some of the molecular strategies organisms use to adjust the membrane lipid composition in response to a decrease in environmental temperature. While the activities of all biomolecules are altered as a function of temperature, the thermosensors we focus on here are molecules whose temperature sensitivity appears to be linked to changes in the biophysical properties of membrane lipids. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus.

Spin-label electron spin resonance (ESR) methods have been used to study the structure of the envelope of vesicular stomatitis virus (VSV). The data indicate that the lipid is organized in a bilayer structure. Proteolytic digestion of the glycoproteins which are the spike-like projections on the outer surface of the virus particle increases the fluidity of the lipid bilayer. Since the lipid composition of the virion reflects the composition of the host plasma membrane and the protein composition is determined by the viral genome, VSV was grown in both MDBK and BHK21-F cells to determine the effect of a change in lipid composition on the structure of the lipid bilayer of VSV. The lipid bilayer of the virion was found to be more rigid when derived from MDBK cells than from BHK21-F cells. Studies comparing spin-labeled intact cells and cell membrane fractions suggest that upon labeling the whole cell the spin label probes the plasma membrane. Comparison of spin-labeled VSV particles and their host cells indicates that the lipid bilayer of the plasma membrane is considerably more fluid than that of the virion. These results are discussed in terms of the effect of membrane-associated protein on the structure of the lipid bilayer.     

Membrane organization and lipid rafts

Cell membranes are composed of a lipid bilayer, containing proteins that span the bilayer and/or interact with the lipids on either side of the two leaflets. Although recent advances in lipid analytics show that membranes in eukaryotic cells contain hundreds of different lipid species, the function of this lipid diversity remains enigmatic. The basic structure of cell membranes is the lipid bilayer, composed of two apposing leaflets, forming a two-dimensional liquid with fascinating properties designed to perform the functions cells require. To coordinate these functions, the bilayer has evolved the propensity to segregate its constituents laterally. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. This principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to focus and regulate membrane bioactivity. Here we will review the emerging principles of membrane architecture with special emphasis on lipid organization and domain formation.     

Sphingomyelin composition and physical asymmetries in native acetylcholine receptor-rich membranes

Selective enzymatic hydrolysis, lipid compositional analyses, and fluorescence studies have been carried out on acetylcholine receptor (AChR)-rich membranes from Torpedinidae to investigate the topology of sphingomyelin (SM) in the native membrane and its relationship with the AChR protein. Controlled sphingomyelinase hydrolysis of native membranes showed that SM is predominantly (approximately 60%) localized in the outer half of the lipid bilayer. Differences were also observed in the distribution of SM fatty acid molecular species in the two bilayer leaflets. A fluorescent SM derivative ( N-[10-(1-pyrenyl)decanoyl]sphingomyelin; Py-SM) was used to study protein-lipid interactions in the AChR-rich membrane and in affinity-purified Torpedo AChR reconstituted in liposomes made from Torpedo electrocyte lipid extracts. The efficiency of F?rster resonance energy transfer (FRET) from the protein to the pyrenyl-labeled lipid as a function of acceptor surface density was used to estimate distances and topography of the SM derivative relative to the protein. The dynamics of the lipid acyl chains were explored by measuring the thermal dependence of Py-SM excimer formation, sensitive to the fluidity of the membrane. Differences were observed in the concentration dependence of excimer/monomer pyrenyl fluorescence when measured by direct excitation of the probe as against under FRET conditions, indicating differences in the intermolecular collisional frequency of the fluorophores between bulk and protein-vicinal lipid environments, respectively. Py-SM exhibited a moderate selectivity for the protein-vicinal lipid domain, with a calculated relative affinity K(r) approximately 0.55. Upon sphingomyelinase digestion of the membrane, FRET efficiency increased by about 50%, indicating that the resulting pyrenyl-ceramide species have higher affinity for the protein than the parental SM derivative.     

Fluorescence energy transfer in two dimensions. A numeric solution for random and nonrandom distributions.

A method of Monte Carlo calculations has been applied to the problem of fluorescence energy transfer in two dimensions in order to provide a quantitative measure of the effects of nonideal mixing of lipid and protein molecules on the quenching profiles of membrane systems. These numerical techniques permit the formulation of a detailed set of equations that describes in a precise manner the quenching and depolarization properties of planar donor-acceptor distributions as a function of specific spectroscopic and organizational parameters. Because of the exact nature of the present numeric method, these results are used to evaluate critically the validity of previous approximate treatments existing in the literature. This method is also used to examine the effects of excluded volume interactions and distinct lattice structures on the expected transfer efficiencies. As a specific application, representative quenching profiles for protein-lipid mixtures, in which donor groups are covalently linked to the protein molecules and acceptor species are randomly distributed within lipid domains, have been obtained. It is found that the existence of phase-separated protein domains gives rise to a shielding effect that significantly decreases the transfer efficiencies with respect to those expected for an ideal distribution of protein molecules. The results from the present numerical study indicate that the experimental application of fluorescence energy transfer measurements in multicomponent membrane systems can be used to obtain organizational parameters that accurately reflect the lateral distribution of protein and lipid molecules within the bilayer membrane.     

Formation of lipid raft nanodomains in homogeneous ternary lipid mixture of POPC/DPSM/cholesterol: Theoretical insights

Lipid rafts, in biological membranes, are cholesterol-rich nanodomains that regulate many protein activities and cellular processes. Understanding the formation of the lipid-raft nanodomains helps us elucidate many complex interactions in the cell. In this study, the formation of lipid-raft nanodomains in a ternary palmitoyl-oleoyl-phosphatidylcholine/stearoyl-sphingomyelin/cholesterol (POPC/DPSM/Chol) lipid mixture, the most realistic surrogate model for biological membranes, has been successfully observed for the first time in-silico using microsecond timescale molecular dynamics simulations. The model reveals the formation of cholesterol-induced nanodomains with raft-like characteristics and their underlying mechanism: the cholesterol molecules segregate themselves into cholesterol nanodomains and then enrich the cholesterol-rich domain with sphingomyelin molecules to form a lipid raft thanks to the weak bonding of cholesterol with sphingomyelin. Besides, it is found that the increase in cholesterol concentration enhances the biophysical properties (e.g., bilayer thickness, area per lipid headgroup, and order parameter) of the lipid raft nanodomains. Such findings suggest that the POPC/DPSM/Chol bilayer is a suitable model to fundamentally extend the nanodomain evolution to investigate their lifetime and kinetics as well as to study protein-lipid interaction, protein-protein interaction, and selection of therapeutic molecules in the presence of lipid rafts.